Polyunsaturated fatty acids (PUFAs) plays an important role in the organism as a constituent of the cell membrane phosphatide, as well as a precursor of the hormone-like physiologically active substance such as prostagrandin, thromboxane, leukotriene and the like. The physiologically active substance, which is synthesized from PUFAs, is called eicosanoid. Eicosanoid is synthesized as needed in the body, and regulates inflammatory reaction, reproduction function, immunological response, blood pressure and the like. In addition as for PUFAs, it has been reported that it is necessary for the cerebral development of the infant.
The PUFAs is classified into series called n-3, n-6, n-9 and the like along the route of biosyntheses. Herein, the numeral “3”, “6”, and “9” following “n-” shows in which number of carbon the first double bond is present from the methyl group of PUFAs. For example, the “n-3” series shows PUFAs, wherein the first double bond from the methyl group is present in the third carbon when the carbon of the methyl group is made a carbon in first position, and it is assumed second position, third position and so on, one by one toward the carboxyl group side, and is displayed as ω3 series. In FIG. 7, major n-3 and n-6 series PUFAs in animal are shown. In n-3 (ω3) series, α-linolenic acid, which is an essential fatty acid, stearidonic acid, 20:4Δ8,11,14,17, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), etc., which are a metabolite from α-linolenic acid (ALA) in vivo, are included. Moreover, n-6 (ω6) series includes linoleic acid (LA), which is an essential fatty acid, and γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA or ARA), etc., Which are a metabolite from linoleic acid in vivo. For example, “18:2” in the indication represented by “18:2Δ9,12” shows that the number of carbons is 18 and the number of double bonds is two. Moreover, “Δ9,12” shows the position of double bond when the carbon of the carboxyl group is made a carbon in first position, and it is assumed second position, third position and so on, one by one toward the methyl group. In addition, since animals cannot desaturate C—C bond on methyl group over Δ9 position, they have to take Q-linolenic acid and linoleic acid from food (vegetable food) as an essential fatty acid, and cannot convert n-6 series to n-3, or n-3 series to n-6, interchangeably.
It is well known that n-6 and n-3 series PUFAs function differently. Indeed, both play an important role in the body. For n-3 series PUFAs, it is known that there have a lot of physiological activities commencing with antithrombotic action and improvement of serum lipid for EPA, and improvement of learning function and anticancer action for DHA. The n-3 series PUFAs are essential for maintaining homeostasis. As described above, since the animals cannot synthesize n-3 series PUFAs in vivo, it is very important for them to take n-3 series PUFAs orally.
As mentioned above, to synthesize n-3 series PUFAs, which importance is recently pointed out, ω3 fatty acid desaturase having an activity that generates ω3 unsaturated fatty acid by forming unsaturated bond between the third and the fourth positions from methyl group of fatty acid, i.e., ω3 and ω4 positions, is necessary. The gene for ω3 fatty acid desaturase has been cloned so far in higher plant, green algae, C. elegans, oomycetes, ascomycetes and the like (for example, see Japanese Patent Application Laid-Open (Kokai) No. 2001-95588 (published on Apr. 10, 2001); International Publication W003/064596 (published on Aug. 7, 2003); Science 258: 1353-1355 (1992); Biosci. Biotechnol. Biochem. 66: 1314-1327 (2002); Proc. Natl. Acad. Sci. USA, 94: 1142-1147 (1997); Biochemistry 39: 11948-11954 (2000); Biochem. J. 378: 665-671 (2004); Microbiology 150: 1983-1990 (2004); and Biochem. Biophys. Res. Commun. 150: 335-341 (1988)).
In Japanese Patent Application Laid-Open (Kokai) No. 2001-95588, Science 258: 1353-1355 (1992) and Biosci. Biotechnol. Biochem. 66: 1314-1327 (2002), it has been reported that the protein that is encoded by ω3 fatty acid desaturase gene of higher plants and/or green algae has an activity, which converts n-6 series fatty acid such as linoleic acid (18:2) having 18 carbons to n-3 series such as α-linolenic acid (18:3). However, the protein that is encoded by ω3 fatty acid desaturase gene of higher plants and green algae cannot convert a fatty acid having 20 carbons to n-3 series fatty acid.
In Proc. Natl. Acad. Sci. USA, 94: 1142-1147 (1997) and Biochemistry 39: 11948-11954 (2000), it has been reported that the proteins that is encoded by ω3 fatty acid desaturase gene (FAT-1) of Caenorhabditis elegans (C. elegans) acts on n-6 series fatty acid having 16-20 carbons and generate unsaturated bond at the ω3 position. However, in Biochemistry 39: 11948-11954 (2000), it has been reported that when this ω3 fatty acid desaturase gene is expressed in yeast, the conversion rate from arachidonic acid (20:4), which is n-6 series fatty acid, to eicosapentaenoic acid (EPA) (20:5), which is n-3 series fatty acid, is low, being only 1.9%.
Moreover, in International Publication WO03/064596, Japanese Patent Application Laid-Open (Kokai) No. 2001-95588 and Biochem. J. 378: 665-671 (2004), the protein that is encoded by ω3 fatty acid desaturase gene (SDD17) of oomycetes (Saprolegnia diclina) acts on n-6 series fatty acid having 20 carbons and generates unsaturated bond at the ω3 position. However, on the contrary, it cannot make the ω3 position of n-6 fatty acid having 18 carbons unsaturated.
Moreover, in Microbiology 150: 1983-1990 (2004), it has been reported that ω3 fatty acid desaturase gene was cloned from Saccharomyces kluyveri, which belongs to ascomycetes. The protein that is encoded by this gene has an activity converting linoleic acid (18:2), which is n-6 series, to α-linolenic acid (18:3), which is n-3 series. However, on the contrary, the ω3 fatty acid desaturase cannot make the ω3 position of n-6 fatty acid having 20 carbons unsaturated.
Meanwhile, it is known that when Mortierella alpina, which is a lipid production fungi, is stood in the condition of low temperature, it generates eicosapentaenoic acid (EPA). That is, eicosapentaenoic acid is not generated when M. alpina is cultured at 25° C., but generated when cultured at 11° C. using glucose as a carbon source (see, for example, Biochem. Biophys. Res. Commun. 150: 335-341 (1988)). From this result, it is suggested that there is ω3 fatty acid desaturase, wherein its gene expression is induced or activated under condition of low temperature.
Moreover, it is known that eicosapentaenoic acid is accumulated in the fungus cells when many strain of Mortierella subgenera is cultured at the low temperature. In that case, since no n-3 series fatty acid other than eicosapentaenoic acid (EPA) was detected, it was considered that eicosapentaenoic acid (EPA) was generated by unsaturation of ω3 position of arachidonic acid under the low temperature condition (see J Am Oil Chem Soc 65, 1455-1459 (1988)). From this result, it is strongly suggested that ω3 fatty acid desaturase, which makes fatty acids having 20 carbons unsaturated, exists.